IP Library › Granted Patent US 12,252,732
Granted Patent B2
US 12,252,732 · App. 18/339,082 · Granted Mar 18, 2025

Methods and systems for nucleic acid preparation and chromatin analysis

Inventor: Geoffrey McDermott (Livermore, CA)
Assignee: 10x Genomics, Inc.
C12Q1/6806
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Quick Facts
Patent No.
US 12,252,732
App. No.
18/339,082
Granted
Mar 18, 2025
Kind
B2
Abstract

The present disclosure provides methods and systems for nucleic acid preparation and/or analysis. Nucleic acids may be derived from one or more cells. Nucleic acid preparation may comprise generating nucleic acid molecules of varying lengths. Nucleic acid analysis may comprise identifying nucleic acid sequence information with nucleosome position information.

Claims (25)

1. A method comprising:

(a) lysing a biological particle comprising chromatin comprising a native structure that includes a plurality of nucleosomes and one or more accessible regions positioned between the plurality of nucleosomes, wherein the lysing preserves the native structure of the chromatin;

(b) generating a plurality of template deoxyribonucleic acid (DNA) molecules from the one or more accessible regions of the native structure of the chromatin using a plurality of transposon-nucleic acid complexes;

(c) generating a plurality of ribonucleic acid (RNA) molecules from the plurality of template DNA molecules such that the plurality of RNA molecules corresponds to a plurality of positions of the plurality of nucleosomes of the chromatin; and

(d) generating a plurality of complementary DNA (cDNA) molecules from the plurality of RNA molecules using a plurality of template switching oligonucleotides such that the plurality of cDNA molecules corresponds to the plurality of positions of the plurality of nucleosomes of the chromatin.

2. The method of claim 1 , wherein each transposon-nucleic acid complex of the plurality of transposon-nucleic acid complexes comprises a transposase molecule and a first nucleic acid molecule.

3. The method of claim 2 , further comprising:

generating, before generating the plurality of template DNA molecules, the plurality of transposon-nucleic acid complexes using a plurality of transposase molecules and a plurality of first nucleic acid molecules.

4. The method of claim 2 , wherein the transposase molecule comprises a Tn5 transposase.

5. The method of claim 2 , wherein the first nucleic acid molecule comprises a promoter region.

6. The method of claim 5 , wherein the promoter region comprises at least one of a T7 promoter sequence, a T3 promoter sequence, or a SP6 promoter sequence.

7. The method of claim 5 , wherein each RNA molecule of the plurality of RNA molecules is generated such that each RNA molecule comprises the promotor region.

8. The method of claim 2 , wherein the first nucleic acid sequence comprises a first sequencing adaptor region.

9. The method of claim 8 , wherein each RNA molecule of the plurality of RNA molecules is generated such that each RNA molecule comprises the first sequencing adaptor region.

10. The method of claim 9 , wherein each template switching oligonucleotide of the plurality of template switching oligonucleotides comprises a second sequencing adaptor region.

11. The method of claim 10 , wherein each cDNA molecule of the plurality of cDNA molecules is generated such that each cDNA molecule comprises the first sequencing adaptor region and the second sequencing adaptor region.

12. The method of claim 1 , wherein the plurality of RNA molecules includes at least two RNA molecules having different lengths.

13. The method of claim 12 , wherein the plurality of cDNA molecules generated from the plurality of RNA molecules includes at least two cDNA molecules that preserve the different lengths of the at least two RNA molecules of the plurality of RNA molecules.

14. The method of claim 1 , wherein the method is performed in a partition.

15. The method of claim 14 , wherein the partition comprises a droplet.

16. The method of claim 1 , wherein generating the plurality of RNA molecules comprises generating the plurality of RNA molecules using RNA polymerase.

17. The method of claim 1 , wherein each template DNA molecule of the plurality of template DNA molecules is double stranded.

18. The method of claim 1 , wherein each cDNA molecule of the plurality of cDNA molecules is single stranded.

19. The method of claim 1 , wherein lysing the biological particle comprises lysing the biological particle using a lysis reagent that preserves chromatin structures.

20. The method of claim 1 , wherein the biological particle is a single cell or a single cell nucleus.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 8, 2023
From: MCDERMOTT, GEOFFREY
To: 10X GENOMICS, INC.
Reel/Frame 065498/0362 →
Continuity (4)
Continuation 16854475 · Apr 21, 2020
Continuation PCTUS2018057156 · Oct 23, 2018
Provisional Application 62577447 · Oct 26, 2017
Related Publication 20240052397A1 · Feb 15, 2024
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